The Na, K and H, K-ATPases are members of the P-type family of ion pumps and play critical roles in maintaining cellular homeostasis and transepithelial transport. The Na, K-ATPase creates ion gradients that maintain cellular osmotic balance and membrane potential. These gradients are exploited by transporting epithelia to drive the import and export of a wide range of solutes against steep concentration gradients. The H, K-ATPase is responsible for gastric acid secretion and also appears to play a role in renal potassium reabsorption. To carry out these functions, these pumps must be restricted to specific domains of the plasma membranes of polarized epithelial cells. The Na, K-ATPase resides at the basolateral surfaces of most polarized epithelial cell types. In the acid secreting parietal cells of the stomach the H, K-ATPase is stored in an intracellular vesicular compartment that fuses with apical plasma membrane in response to secretagogue stimulation. The activities of these ion pumps are tightly controlled through a variety of processes that regulate both their subcellular trafficking and their catalytic properties. In order to attain their appropriate subcellular distributions and to participate in their respective regulatory pathways, both pumps are likely to interact with a large number of accessory proteins. During the previous funding period we have identified a several novel partner polypeptides that appear to interact with these pumps to modulate their cell biologic and functional properties. We find that the Na,K-ATPase associates with both neurabin II/spinophilin and arrestin, suggesting that the sodium pump may be susceptible to regulatory mechanisms similar to those that govern G protein coupled receptor signaling. In addition, the Na, K and H, K-ATPase each form complexes with a member of the tetraspan family of transmembrane interacting proteins, and these associations profoundly influence pump trafficking. In the present proposal we will carry out studies designed to 1) determine the molecular and cell biologic mechanisms through which neurabin II/spinophilin and arrestin modulate the function of the Na, KATPase, 2) characterize the physiologic effects of interactions between ion pumps and tetraspans, and 3) examine the role of interacting proteins in governing ion pump function in vivo. These studies will allow us to define the physiological significance of these novel interactions, and to understand their involvement in governing pump function both under normal circumstances and in the context of pathological conditions.